Development of Standard and Competitive ELISA for Detection and Quantification of PRP-1 in Biological Fluids by Using anti-PRP-1 Polyclonal Antiserum

ORIGINAL ARTICLE
N. V. Tumasyan, I. K. Sahakyan, N. V. Kocharyan, A. A. Khachatryan, T. K. Davtyan, K. A. Galoyan, S. S. Abrahamyan

 
For citation: Tumasyan NV, Sahakyan IK, Kocharyan NV, Khachatryan AA, Davtyan TK, Galoyan KA, Abrahamyan SS. Development of Standard and Competitive ELISA for Detection and Quantification of PRP-1 in Biological Fluids by Using anti-PRP-1 Polyclonal Antiserum. International Journal of Biomedicine. 2024;14(3):516-519. doi:10.21103/Article14(3)_OA21
 
Originally published September 6, 2024

Abstract: 

This study aimed to establish a sensitive method for the detection of proline-rich polypeptide-1 (PRP-1) in biological fluids. PRP-1, also known as galarmin, is a fragment of neurophysin-vasopressin-associated glycoprotein synthesized by brain neurosecretory cells and consisting of 15 amino acid residues. An enzyme-linked immunosorbent assay (ELISA) was used for PRP-1 quantification. An ELISA system has been developed using polyclonal antibodies we raised against the synthetic PRP-1. According to the analysis, the concentration PRP-1 of 25 ng/mL was accepted as the main appropriate coating concentration for further experiments in 1:100 and 1:500 antibody dilutions. Then, a competitive ELISA was developed to quantify PRP-1 in the fluids.
Based on the results, an appropriate condition was chosen to be the best condition for PRP-1 detection: the appropriate quantity of the immobilized PRP-1 (25 ng/mL); anti-rabbit primary antibodies against PRP-1 (1:500); anti-rabbit secondary antibodies conjugated to peroxidase (1:1000), and extravidin (1:1000); as a result, the minimum detectable amount of PRP-1 in the fluid was 1.5 ng/mL. Thus, this method provides a good detection limit and sensitivity and is easy to use. In addition, a large number of rats and human serum and plasma samples can be analyzed rapidly and simultaneously, which is what we intend to realize in the future.

Keywords: 
PRP-1 • galarmin • ELISA
References: 
  1. Galoyan AA. Biochemistry of Novel Cardioactive Hormones and Immunomodulators of the Functional System Neurosecretory Hypothalamus – Endocrine Heart. Nauka Publishers, Moscow; 1997, 242 pp.
  2. Galoyan AA, Sarkissian JS, Kipriyan TK, Sarkissian EJ, Chavushyan EA, Sulkhanyan RM, Meliksetyan IB, Abrahamyan SS, Grigorian YKh, Avetisyan ZA, Otieva NA. Protective effect of a new hypothalamic peptide against cobra venom and trauma-induced neuronal injury. Neurochem Res. 2001 Sep;26(8-9):1023-38. doi: 10.1023/a:1012353005489. PMID: 11699930.
  3. Galoyan AA, Sarkissian JHS, Chavushyan VA, et al. Study of the newhypothalamic Proline-rich peptide (PRP-1) protective effect on morpho-functional changes in rat hippocampus using a model of Alzheimer’s Disease induced by intracerebroventricular injection of Beta-Amyloid peptide Aβ (25-35). J. Neurochemistry. 2004;21(4):265-288.
  4. Abrahamyan SS, Sarkissian JS, Meliksetyan IB, Galoyan AA. Survival of trauma-injured neurons in rat brain by treatment with proline-rich peptide (PRP-1): an immunohistochemical study. Neurochem Res. 2004 Apr;29(4):695-708. doi: 10.1023/b:nere.0000018840.19073.0b. PMID: 15098931.
  5. Abrahamyan SS, Meliksetyan IB, Chavushyan VA, Aloyan ML, Sarkissian JS. Protective action of snake venom Naja naja oxiana at spinal cord hemisection. Ideggyogy Sz. 2007 Mar 30;60(3-4):148-53. PMID: 17451057.
  6. Engvall E. Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 1980;70(A):419-39. doi: 10.1016/s0076-6879(80)70067-8. PMID: 6999296.

Download Article
Received July 15, 2024.
Accepted August 23, 2024.
©2024 International Medical Research and Development Corporation.